ABSTRACT: Extracellular vesicles (EVs) released by cells contain mRNAs, miRNAs, lncRNAs, lipids, and proteins, playing crucial roles in cell-cell communication. While full-length mRNA transcripts have been documented in EVs secreted by cancer cells, there are no reports on full transcripts secreted by embryos. Our study aimed to identify extracellular vesicle mRNAs in the culture media of bovine embryos and investigate their roles in embryo-mother communication. Following the isolation of EVs from in-vitro fertilization media samples and RNA sequencing, we identified full mRNA transcripts of three genes: SLBP2, ACCSL, and DPPA3. We selected DPPA3 for further investigation. To examine the role of DPPA3 in embryo-mother communication, an in vitro transcribed mRNA of DPPA3 was transfected into bovine endometrial epithelial cells. Transfected and control cells were subsequently analyzed for RNA sequencing to assess the effects of DPPA3 on gene expression. A total of 24 genes were found to be upregulated, and one gene was downregulated (FDR < 0.01) following DPPA3 transfection. Among the 18 annotated genes, 17 have known functions in pregnancy recognition in ruminants or have been previously identified as upregulated in pregnant animals. In addition to RNA sequencing, transfected and control cells underwent proteomic analysis. A total of 34 proteins were differentially expressed (FDR < 0.01, p < 0.05, fold change > 1.5), with 19 upregulated and 15 downregulated. Two proteins, ISG15 and MX1, overlapped with the differentially expressed mRNAs. To mimic the natural transfer of EVs from embryos to endometrial cells, we performed co-culture of day 8 blastocysts or supplemented the cells with embryo-conditioned culture media. DPPA3 expression was detected in endometrial cells exposed to embryo-conditioned media after just 30 minutes, with slight upregulation of ISG15 and MX1 also observed. The expression of these classic interferon-stimulated genes in endometrial cells increased from 0.5 to 10 hours when exposed to blastocysts or embryo-conditioned media. Overall, these results demonstrate that DPPA3 mRNA secreted from embryos may play a role in early embryo-mother communication and maternal recognition of pregnancy prior to major interferon tau secretion. Importantly, our study highlights the significant role of EVs in cell-cell communication through mRNA signaling from the embryo to the mother.
